The inhibition of dopamine reuptake via the dopamine transporter (DAT) has been characterized as the primary mechanism by which cocaine produces its psychomotor stimulant and reinforcing actions. In order to understand further the molecular mechanisms underlying cocaine abuse, structure-function studies have been directed toward characterizing the DAT protein at a molecular level. The design, synthesis and evaluation of 3-alpha-(diphenylmethoxy)tropane (benztropine, BZT) analogs have provided potent and selective probes for the DAT. Structure-activity relationships (SAR) have been developed that contrast with those described for cocaine, despite significant structural similarity. Furthermore, behavioral evaluation of many of the BZT analogs, in animal models of cocaine abuse, has suggested that these two classes of tropane-based dopamine uptake inhibitors have distinct pharmacological profiles. In general, our previous studies have shown that the BZT analogs, do not demonstrate efficacious locomotor stimulation in mice, do not fully substitute for a cocaine discriminative stimulus and are not appreciably self-administered in rats, rhesus or squirrel monkeys. These compounds are generally more potent than cocaine as dopamine uptake inhibitors, in vitro, although their actions in vivo are not consistent with this action. By varying the structures of the parent compounds and thereby modifying their physical properties, pharmacokinetics (PK) as well as pharmacodynamics (PD) is directly affected. Evaluating these compounds in both in vitro and in vivo models to obtain PK and PD profiles on these agents, in comparison to cocaine, with a series of N-substituted BZT analogues has demonstrated that these compounds readily penetrate the blood brain barrier, but compared to cocaine, they have a slower onset and duration of action, which is a suitable profile for development as pharmacotherapeutics and may be directly related to their lack of cocaine-like behavioral profiles. Further investigation into correlating structure, pharmacological action and PD of this class of compounds and developing these agents as potential cocaine-abuse therapeutics is ongoing. In this regard, we have extended the studies on our previously characterized cocaine antagonist, JHW 007 (N-n-butyl-4, 4-diF-BZT), and discovered that JHW 007 and structurally related benztropine analogues were not self administered, in rats and indeed could selectively attenuate self administration of cocaine while having no effect on food administration. These studies further demonstrate the lack of abuse liability for these agents and strengthen their potential for development as therapeutics. Further evaluation of these and other N-substituted BZTs using microdialysis has allowed us to relate the rate and levels of increasing extracellular dopamine, in vivo, with binding of these compounds to the DAT. Further investigation has shown that several additional analogues of JHW007 show similar profiles in vivo and have been identified as lead candidates for development as medications to treat cocaine abuse, as well as attention deficit hyperactivity disorder (ADHD). Recent studies using site-directed mutagenesis have revealed differences in binding domains between the BZTs, cocaine and other structurally diverse dopamine uptake inhibitors. Interestingly, experimental evidence using the DAT inhibitors cocaine, WIN 35,428, and several benztropine analogues and comparing them to the substrates dopamine and MDMA has provided evidence, at the molecular level, of binding interaction differences that correlate with their distinctive behavioral profiles. Further synthetic studies are focused on novel 2-substituted BZT and benztropinamines analogues that incorporate oxazole functionality into the parent structure. These compounds will be characterized in vitro, in due course. In addition to developing agents for in vivo studies, we have also synthesized a number of important molecular tools in the form of fluorescent-derivatives of our tropane based DAT inhibitors. One such compound, JHC 1-064, has been used to characterize the trafficking and cellular distribution of DAT in living neuronal cells. Indeed, recent experiments in living neurons with JHC 1-064 have provided data that challenge mechanistic dogma for transporter translocation, as determined in DAT transfected heterologous cells, which is one area of ongoing research with these agents. Further, JHC 1-064 binds with high affinity to the serotonin transporter (SERT), as well, and we are conducting analogous studies, first in cell lines to study trafficking and cellular distribution of SERT. This project is related to a new project in the laboratory in which we have designed and synthesized novel analogues of the SERT inhibitor and antidepressant, citalopram. Citalopram is a racemic mixture of (+)- and (-) enantiomers, of which the latter (S-(+)-citalopram) is the more active antidepressant and currently marketed as Escitalopram. However, the (-)-enantiomer also binds to the SERT, and evidence using site directed mutagenesis, suggests that these enantiomers may be accessing different binding sites. Further these data suggest that the (-)-enantiomer is binding to an allosteric site that can affect the binding of the (+)-enantiomer and may be responsible for some diminution of its actions in vivo. As such, we have designed and synthesized several series of citalopram analogues and separated several sets of enantiomers to further characterize and compare binding profiles at both the SERT and the other monoamine transporters, with the parent ligand. In addition, behavioral studies are being conducted with selected analogues using drug discrimination in rats. Our goal is to ultimately relate structure of the citalopram analogues to behavior and further to characterize the SERT binding interactions at the molecular level, to further understand and characterize the role of this putative allosteric site.